NEUROTROPHIN RECEPTOR P75NTR AND ITS LIGAND BDNF IN HUMAN SKELETAL MUSCLE:INVOLVEMENT IN MYOGENESIS AND MUSCLE REPAIR

Neurotrophins are growth factors fundamental for the development and the maintenance of the central nervous system. Recent studies in animal models highlighted a role for neurotrophins and neurotrophin receptors in skeletal muscle biology. However, little is known about their expression in human muscle. The aim of this thesis was to study the expression of the neurotrophin receptor p75NTR in human skeletal muscle, and to investigate its involvement in myogenesis and in muscle regeneration. Then this work focused on the ligands that trigger p75NTR signalling. We combined molecular and cellular analyses of primary muscle precursor cells with histological investigations of muscle biopsies from non myopathic specimens, and from patients affected by inflammatory myopathies and Becker muscular dystrophy. In physiological conditions p75NTR was displayed by the great majority of satellite cells in vivo and it was present on regenerating fibers in inflamed and dystrophic muscle. In vitro experiments, such as receptor blockade and gene silencing in myoblasts, demonstrated that it was necessary for differentiation of muscle precursor cells and regulation of dystrophin induction in myotubes. Transcriptome analyses of p75NTRhigh vs. p75NTRlow muscle cells showed that p75NTR is the prototype marker for a precursor cell population bearing a transcriptional repertoire associated with muscle development and maturation. Molecular and immunohistochemical experiments highlighted enhanced p75NTR expression in all the inflammatory myopathies but not in Becker muscular dystrophy. In inflamed muscle p75NTR was localised on distinct cell types, including immune cells and mature myofibers. In vitro assays on human myotubes confirmed that inflammatory factors such as IL-1 could induce p75NTR. RNA interference in differentiated cells demonstrated that the neurotrophin receptor was fundamental for myofiber survival under inflammatory stress. Interestingly, we recorded a reduction in the pool of p75NTR expressing satellite cells in inflammatory myopathy biopsies. Regarding p75NTR ligands, we found that BDNF was the major neurotrophin expressed by precursor and differentiated cells in vitro. Accordingly, it was displayed by muscle satellite cells and mature myofibers in vivo. Differential analysis of BDNF receptors showed concomitant expression of p75NTR, suggesting that the BDNF-p75NTR axis is predominant in skeletal muscle cells. Several functional experiments in vitro demonstrated that BDNF gene silencing or protein blockade in myoblast cultures hampered myogenesis. Finally, histological investigations of inflammatory myopathy biopsies, revealed that infiltrating immune cells localized preferentially near p75NTR positive regenerating fibers and that they produced BDNF, suggesting that both muscle and immune cell derived BDNF is available to support the regeneration of p75NTR expressing fibers. In conclusion, these data demonstrate that p75NTR and BDNF are profoundly involved in human myogenesis and in muscle repair and survival to inflammatory stress.